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Hydrogen sulfide (H(2)S) is a volatile gas of considerable interest as a physiologically relevant signaling molecule, but this volatility has typically been overlooked in the context of biological experiments. We examined volatility of 10 and 100 μM H(2)S (Na(2)S·9H(2)O) in real time with polarographic electrodes in three commonly employed experimental apparatuses: 24-well tissue culture plates (WP), muscle myograph baths (MB), and the Langendorff perfused heart apparatus (LPH). H(2)S loss from all apparatuses was rapid and exponential, with half-times (t(1/2)) of 5 min (WP), less than 4 min (MB), and less than 0.5 min (LPH). The t(1/2) for H(2)S loss from MB bubbled with 100% oxygen was slightly longer than that for MB bubbled with 100% nitrogen; both were significantly shorter than stirred but unbubbled MB (9 min). Therefore, even without tissue, H(2)S rapidly disappears from buffer under a variety of experimental conditions, and this is due to volatilization, not oxidation. The inability to maintain H(2)S concentration, even briefly, questions the accuracy of dose-response studies and the relevance of long-term (10 min) exposure to a single treatment of H(2)S. These results also help to explain the discrepancy between low H(2)S concentrations in blood and tissues versus high concentrations of exogenous H(2)S required to produce physiological responses.

“Sulfhydration” has been proposed to be a means of hydrogen sulfide (H 2 S) signaling in which the H 2 S reacts with the sulfur of a cysteine residue in proteins, thereby forming a polysulfide and changing the function of the latter, as described by the equation RSH + H 2 S → RSSH. However, this requires oxidation of the cysteine RSH, which cannot be accomplished by H 2 S, as the sulfur is in its most reduced form (oxidation state of −2). Hydropersulfides (H 2 S n , n = 2–8, oxidation number = 0), can oxidize RSH and have been suggested to be a contaminant of salts commonly used to produce H 2 S (NaSH, Na 2 S) and account for much of the activity originally attributed to H 2 S. Here, we use the green fluorescent protein, roGFP2, that contains two reactive cysteine residues (Cys147, Cys204) and is a specific reporter of redox status, being fully oxidized by 30 mM tert-butyl hydroperoxide (t-BuOOH), and fully reduced by dithiothreitol (DTT, 30 mM), to determine if H 2 S can spontaneously form hydro-persulfides in normoxic (21% O 2 ) laboratory conditions. We show that all concentrations ranging from 1 μ M-300uM H 2 S have very little reductive effect on roGFP, but that H 2 S > 300 uM slowly reoxidizes roGFP in a closed system given an hour to react. We attribute this to spontaneous oxidation of H 2 S to hydropersulfides. Addition of mixed persulfides (K 2 S n , n = 1–8) oxidizes roGFP at low concentrations (1uM-300uM), supporting our hypothesis. These results show that H 2 S n can be formed from H 2 S and thereby signal via sulfhydration. Support, NSF IOS-1051627 and NSF DGE-1313583.